Med Oncol (2015) 32:24 DOI 10.1007/s12032-014-0470-1

ORIGINAL PAPER

Sorafenib reverses resistance of gastric cancer to treatment by cisplatin through down-regulating MDR1 expression Yi-sheng Huang • Zhi Xue • Hua Zhang

Received: 15 December 2014 / Accepted: 17 December 2014 Ó Springer Science+Business Media New York 2015

Abstract Cisplatin (DDP) has been successfully used in the treatment of gastric cancer; however, resistance of gastric tumors to cisplatin has also resulted in frequent treatment failure. To investigate the effect of sorafenib in reversing the resistance of human gastric cancer cell line SGC7901/DDP to treatment by cisplatin, and to examine possible underlying mechanisms. A SGC7901/DDP cell line was treated with different concentrations of sorafenib, with and without cisplatin. The reversing ability of sorafenib on cisplatin treatment was examined using MTT, FACS, and xenograft models. Western blotting and immunofluorescence were used to determine expressions of MDR1, p-Akt, and p-ERK. Sorafenib inhibited proliferation of human gastric cancer cell line SGC7901/DDP, both when used alone and in combination with cisplatin. Expression levels of MDR1, p-Akt, and p-ERK were significantly decreased after sorafenib treatment. Sorafenib may reverse resistance of human gastric cancer cell line SGC7901/DDP to cisplatin through down-regulating MDR1 expression.

Y. Huang Division 1 of Pulmonary Oncology, Guangdong Lung Cancer Institute, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou, China Z. Xue Department of Medical Research, Guangdong General Hospital, Guangdong Lung Cancer Institute, Guangdong Key Laboratory of Lung Cancer Translational Medicine, Guangzhou, China H. Zhang (&) Department of Medical Oncology, Cancer Center, Guangdong General Hospital and Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong, China e-mail: [email protected]

Keywords Stomach neoplasm  Cell line  Drug resistance  MDR  Sorafenib

Introduction Gastric cancer, the fourth most commonly diagnosed cancer worldwide, ranks second in global cancer mortality [1]. The outcome among patients with advanced gastric cancer (AGC) is poor, and systematic chemotherapy plays a critical role in treatment. Cisplatin (DDP) has been commonly used in the treatment of gastric cancer; however, the acquisition of cisplatin resistance is a major clinical obstacle to successful treatment. Therefore, an understanding of the molecular and cellular mechanisms involved in cisplatin resistance is necessary for the development of new and targeted therapeutic strategies and is indispensable for the development of effective chemotherapeutic agents. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) cascade is a key intracellular signaling pathway that regulates diverse cellular functions, including proliferation, survival, apoptosis, motility, transcription, metabolism, and differentiation [2], [3]. The MAPK pathway plays a well-defined role in cancer biology and has been an important target in the development of targeted therapies [4]. Inactivation of the Ras/Raf/MEK/ERK MAPK pathway has been shown to prevent cell proliferation, migration, adhesion, colony formation, replication, and apoptosis. Sorafenib (Nexavar, Bayer), an RAF family kinase inhibitor, is a multikinase inhibitor originally developed as an inhibitor of RAF-1, a component of the extracellular signal regulated kinase (ERK)1/2 pathway, but that was subsequently shown to inhibit numerous other kinases,

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including class III tyrosine kinase receptors such as platelet-derived growth factor (PDGF), VEGF receptors 1 and 2, c-Kit, and FLT3. The antitumor effects of sorafenib in renal cell carcinoma and in hepatoma have been ascribed to antiangiogenic actions, through inhibition of growth factor receptors [5–9]. Based on these findings, we tested whether co-treatment with cisplatin and sorafenib was more effective in the inhibition of cancer cell proliferation than was cisplatin alone. Our results showed that sorafenib sensitizes human cells to cisplatin through down-regulating MDR1 expression.

Materials and methods Cell lines Human gastric adenocarcinoma SGC7901 and cisplatinresistant human gastric adenocarcinoma SGC7901/DDP cell lines were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Reagents and medicines The RPMI 1,640 cell culture solution was purchased from Hyclone (Logan, Utah, USA). Fetal calf serum was purchased from PAA Laboratories (Co¨lbe, Germany). Cisplatin was purchased from Jinan Qilu-Pharma (Shandong, China). Sorafenib was obtained from Bayer (Germany). Methyl thiazolyl tetrazolium (MTT), dimethyl sulfoxide (DMSO), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospho-AKT and phospho-ERK1/2 antibodies were purchased from CellSignaling Technology. The MDR, AKT, ERK1/2, and bactin antibodies were purchased from Santa-Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxide labeling secondary antibody was purchased from Wuhan Boster (Hubei, China). Fluorescence labeling secondary antibody was purchased from Invitrogen. Experimental animals BALB/C nu/nu female nude mice were obtained from the Experimental Animal Center, Guangzhou University of Chinese Medicine (Guangzhou, China).

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serum, at 37 °C and under 5 % CO2 by conventional cell passage. The SGC7901/DDP cell culture medium contained a final concentration of 0.1 mg/L cisplatin. MTT assay Cells at a logarithmic growth period were inoculated into 96-well plates at a density of 5 9 103 per well for 24 h, and then supplemented with treatment agents. Five duplicate samples were analyzed for each medication dose. At 72 h after treatment, 10 lL MTT (5 mg/mL) was added to each well. The culture was terminated 4 h later. Supernatants were abandoned, 150 lL DMSO was added to each well, and the plates were shaken in a dark room for 10 min, allowing for full dissolution of crystalline substances. The optical density (OD) of each well was measured using a microplate reader. The inhibition rate of cell proliferation (%) was calculated as 100 % 9 [(OD value in the control group)-(OD value in the experimental group)]/(OD value in the control group). Apoptosis assay Cells collected during a logarithmic growth period were inoculated into 6-well plates for 24 h and treated with sorafenib for 24 h. The treated cells were collected and adjusted to a cell concentration of 1 9 106/mL. A 100-lL portion of cell suspension was added to a 5-mL tube, to which was added 5 lL Alexa Fluor 488 Annexin V and 5 lL propidium iodide (PI) (20 lg/mL); the mixture was evenly mixed and incubated in the dark for 30 min. Cell apoptosis was determined by flow cytometry fluorescenceactivating cell sorting (FACS). Western blot The target protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane, incubated with target primary antibody after blocking at 4 °C overnight, incubated with corresponding secondary antibody for 1 h after irrigation, treated with ECL reagent, and exposed to film to visualize bands of interest. Detailed experimental procedures were consistent with those of previous investigations [7]. Immunofluorescence assay

Experimental methods Cell cultures The SGC7901 and SGC7901/DDP cell lines were cultured in RPMI1640 culture medium containing 10 % fetal calf

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Cells at a logarithmic growth period were inoculated into covered 6-well plates for 24 h and treated with sorafenib for 24 h. The treated cells were collected and fixed in 4 % paraformaldehyde, and subsequently treated with 0.2 % Triton X-100. The cells were subjected to anti-MDR1

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primary antibody incubation for 45 min, secondary antibody incubation for 30 min, and sealed on glass slides with 40 ,6-diamidino-2-phenylindole (DAPI). The fluorescence status was observed under a microscope. Animal experiments Tumor cells were inoculated via a conventional subcutaneous route, and medication was administered after obtaining stable tumors. Changes in tumor size were measured. The experimental procedures have been described in detail in previous studies [8]. Statistical analysis The statistical software SPSS v. 11.15 was employed for data analysis. Mean values were compared using a one-way ANOVA and a least significant difference (LSD)-t test. Interactive effects were analyzed using factorial analysis. Mean values among groups were compared with a one-way ANOVA. Paired comparisons were conducted by LSDs. Heterogeneity of variance was analyzed by Dunnett-T3. Ranking data were statistically analyzed using an independent sample test.

Results The cisplatin-resistant cell line SGC7901/DDP exhibits high expressions of MDR1. An MTT assay revealed a significant difference between the growth-inhibiting effect of cisplatin on normal SGC7901cells and on cisplatinresistant SGC7901/DDP cells (p \ 0.01). The half-maximal inhibitory concentration (IC50) of SGC7901 cells was 0.61 mg/L, versus an IC50 of 9.95 mg/L for the SGC7901/ DDP cells. Western blotting detected a high expression of

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MDR1 in the drug-resistant cells; the expression levels of p-Akt and p-ERK also increased, while that of ERK remained unchanged, as shown in Fig. 1. Sorafenib may induce apoptosis of SGC7901/DDP cells, with the proportion of apoptosis dependent upon drug concentration. The SGC7901/DDP cells were treated with different doses of sorafenib for 24 h, and the proportions of cellular apoptosis were detected by flow cytometry. Results showed that the apoptosis proportion of untreated cells was 1.87 ± 0.32 %, while after treating with 1.5, 3, 6, and 12 lM sorafenib, the apoptosis proportions increased by up to 5.27 ± 0.25, 6.63 ± 0.15, 15.63 ± 0.55, and 54.77 ± 1.08 %, respectively, indicating that the apoptosis proportion is positively correlated with sorafenib concentration (p \ 0.05), as illustrated in Fig. 2. Sorafenib is able to inhibit the expression of MDR in drug-resistant cells, decrease the expression of Akt, and activate the expression of ERK. The SGC7901/DDP cells were treated with different doses of sorafenib for 24 h, and the expression level of target protein was measured by Western blot. Results showed that MDR1 was strongly expressed in drug-resistant cells. In addition, activation levels of p-Akt and p-ERK were enhanced in drug-resistant cells. After treatment with different doses of sorafenib, the expression levels of MDR1, p-Akt, and p-ERK were significantly enhanced. The degree of tumor decline was positively correlated with the sorafenib concentration, whereas levels of Akt and ERK protein showed no such dependence. An immunofluorescence assay was employed to detect the expression of MDR1 in different cells. The expression of MDR1 in normal cells was low, while its expression was significantly enhanced in drug-resistant cells; the enhanced MDR1 expression was not detected after sorafenib treatment, as shown in Fig. 3. Sorafenib can inhibit the growth of SGC7901/DDPtransplanted tumors, and the inhibitory effect can be

Fig. 1 a An MTT assay revealed that the growthinhibiting effect of cisplatin on cisplatin-resistant SGC7901/ DDP cells was significantly different than the effect on normal SGC7901cells (p \ 0.01). b Western blotting detected a high expression of MDR1 in the treatment of drugresistant cells; the expression levels of p-Akt and p-ERK increased, while that of ERK protein remained unchanged

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Discussion

Fig. 2 a SGC7901/DDP cells were treated with different doses of sorafenib for 24 h, and the proportion of cellular apoptosis detected by flow cytometry. b The apoptosis proportion was positively correlated with the concentration of sorafenib (1.5, 3, 6, and 12 lM) used in the treatment (p \ 0.05)

strengthened by use in combination with cisplatin. The nude mouse models with tumors were utilized to analyze the inhibition by sorafenib on SGC7901/DDP-transplanted tumors. After obtaining stable tumors, the animals were divided into a control group, a cisplatin treatment group, a sorafenib treatment group, and a combined sorafenib with cisplatin treatment group, with six mice per group. The results showed that the inhibition of drug-resistant cell tumors by cisplatin was low. The inhibitory effect of sorafenib on tumor growth, on the other hand, was high (p \ 0.05). Moreover, the inhibition effect was significantly enhanced with the combined use of sorafenib and cisplatin, with the effect of combined use being significantly higher than the use of either sorafenib or cisplatin alone, as shown in Fig. 4.

Fig. 3 a SGC7901/DDP cells were treated with different doses of sorafenib for 24 h, and the expression level of target proteins (MDR1, p-Akt, p-ERK, Akt, and ERK) measured by Western blot. b Expressions of MDR1 in different cells were detected by immunofluorescence

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The present study indicates that sorafenib might present a new treatment option for the management of metastatic gastric carcinoma (GC). Specifically, sorafenib, a multityrosine kinase inhibitor (TKI), exhibited cytotoxic effects on different GC cell lines. Moreover, the antitumor activity of cisplatin was more pronounced when it was used in combination with sorafenib, both in vitro and in vivo, showing that sorafenib enhances the activity of cisplatin. In addition, the effect of sorafenib was observed in cisplatinresistant GC cell clones, indicating that sorafenib can restore cisplatin susceptibility. Sorafenib clearly improves the ability of cisplatin to decrease cell proliferation rates and enhance apoptosis. Sorafenib could, thus, be used to overcome resistance to cisplatin-based chemotherapies in GC. Our study showed that sorafenib overcomes resistance to cisplatin mainly through down-regulating the expression of MDR1. The molecular mechanisms underlying the effect of sorafenib on MDR1 is still under investigation; however, MDR expression is strongly associated with failure in the clinical treatment of cancer and heightened expression is correlated with decreased cellular sensitivities to a broad range of chemotherapeutic agents. These diminished sensitivities are generally due to the overexpression of efflux transport protein on the plasma membrane of cancer cells. Three major human ATP-binding cassette (ABC) transporters have been recognized to play a key role in the expression of MDR, including P-glycoprotein (MDR1/ ABCB1), MDR protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) [10]. Sorafenib has been shown to block the function of ABCB1 and other ABC transporters, including ABCC2, ABCC4, and ABCG2 [11, 12]. Hu et al. [12] showed that sorafenib inhibits the ATPase activity of ABCC2 by directly interacting with this ABC transporter. Carloni et al. [13] reported that sorafenib

assay; MDR1 expression was low in normal cells, but was strongly enhanced in drug-resistant cells. MDR1 expression returned to low levels after sorafenib treatment

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Fig. 4 a The nude mouse models with tumors were used to analyze the inhibition effect of sorafenib on SGC7901/DDPtransplanted tumors. The animals were divided into a control group, a cisplatin treatment group, a sorafenib treatment group, and a combined sorafenib–cisplatin treatment group. b Tumor formation curves after the four different types of treatment

decreases the expression level of ABCC2 in some breast cancer cell lines. Taken together, these works suggest that sorafenib might inhibit both the function and the cell surface expression of ABCG2, ultimately leading to enhanced antitumor effects of chemotherapy drugs. Our results suggest that sorafenib acts on MDR1 (ABCB1) expression. Furthermore, we think that MDR1 expression may become a predictive marker for sorafenib response in cisplatin-resistant tumors. Indeed, many studies have shown that MDR1 expression is increased in human carcinoma treated with cisplatin, and patients with cancers that overexpress MDR1 could be good candidates for combined treatment with sorafenib and cisplatin [14–21]. On the other hand, ABCB1 inhibition is clearly not the only mechanism involved in the synergistic effect of combined sorafenib–cisplatin treatment. Indeed, the sorafenib–cisplatin combination was also more effective than cisplatin alone in GC cells. Sorafenib seems to have little effect on two other major ABC transporters, ABCC1 and ABCG2, involved in drug efflux [11]. In addition, Bareford and colleagues found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Some preliminary evidence suggests the efficacy of sorafenib plus capecitabine/cisplatin in the treatment of AGC patients. The objective response rate of a sorafenib–

capecitabine–cisplatin regimen was 62.5 % (10 of 16 patients; 95 % CI 38.8–86.2 %), and the median progression-free survival and overall survival were 10.0 months (95 % CI 7.4–13.8 months) and 14.7 months (95 % CI 12.0–20.0 months), respectively [22]. In another trial, that examined the efficacy and toxicity of combined sorafenib– docetaxel–cisplatin in patients with metastatic or advanced adenocarcinoma of stomach or gastroesophageal, 18 of 44 eligible and treated patients (41, 90 % CI 28–54 %) showed partial responses to the treatment; the median progressionfree survival was 5.8 months (90 % CI 5.4–7.4 months) and the median overall survival was 13.6 months (90 % CI 8.6–16.1 months). The sorafenib–docetaxel–cisplatin combination thus shows an encouraging efficacy profile with tolerable toxicity. Additional studies of sorafenib in combination with chemotherapy are warranted in cases of gastric cancer [23]. Another oral multi-targeted tyrosine kinase inhibitor, sunitinib, has shown insufficient clinical value as a second-line treatment for AGC; however, its role in combination with chemotherapy merits further study [24, 25]. In conclusion, results of this stud show that sorafenib is a promising option for the treatment of cisplatin-resistant GC. The sorafenib–cisplatin combination is not toxic in xenografted mice. Therefore, investigations of the clinical effects of the sorafenib–sorafenib combination in the treatment of GC should be continued.

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Acknowledgments This study was supported by the Grants from Medicine Science Research of Guangdong Province (Nos. A2009036, A2014034) and Science and Technology Projects of Guangdong Province (No. 2012B031800163) and Clinical Medicine Scientific Research Foundation of Wu Jie-ping (No. 320.6700.09043). Conflict of interest

The authors declare no conflict of interest.

References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. 2. Ramos JW. The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. Int J Biochem Cell Biol. 2008;40(12):2707–19. 3. McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, Chang F, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta. 2007;1773(8):1263–84. 4. Friday BB, Adjei AA. Advances in targeting the Ras/Raf/MEK/ Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy. Clin Cancer Res. 2008;14(2):342–6. 5. Yacoub A, Hamed HA, Allegood J, Mitchell C, Spiegel S, Lesniak MS, et al. PERK-dependent regulation of ceramide synthase 6 and thioredoxin play a key role in mda-7/IL-24-induced killing of primary human glioblastoma multiforme cells. Cancer Res. 2010;70:1120–9. 6. Park MA, Yacoub A, Rahmani M, Zhang G, Hart L, Hagan MP, et al. OSU-03012 stimulates PKR-like endoplasmic reticulumdependent increases in 70-kDa heat shock protein expression, attenuating its lethal actions in transformed cells. Mol Pharmacol. 2008;73:1168–84. 7. Walker T, Mitchell C, Park MA, Yacoub A, Graf M, Rahmani M, et al. Sorafenib and vorinostat kill colon cancer cells by CD95dependent and -independent mechanisms. Mol Pharmacol. 2009;76:342–55. 8. Martin AP, Park MA, Mitchell C, Walker T, Rahmani M, Thorburn A, et al. BCL-2 family inhibitors enhance histone deacetylase inhibitor and sorafenib lethality via autophagy and overcome blockade of the extrinsic pathway to facilitate killing. Mol Pharmacol. 2009;76:327–41. 9. Park MA, Reinehr R, Ha¨ussinger D, Voelkel-Johnson C, Ogretmen B, Yacoub A, et al. Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in gastrointestinal tumor cells. Mol Cancer Ther. 2010;9:2220–31. 10. Szaka´cs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer. Nat Rev Drug Discov. 2006;5:219–34. 11. Wei Y, Ma Y, Zhao Q, Ren Z, Li Y, Hou T, et al. New use for an old drug:inhibiting ABCG2 with sorafenib. Mol Cancer Ther. 2012;11:1693–702.

123

12. Hu S, Chen Z, Franke R, Orwick S, Zhao M, Rudek MA, et al. Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters. Clin Cancer Res. 2009;15:6062–9. 13. Carloni S, Fabbri F, Brigliadori G, Ulivi P, Silvestrini R, Amadori D, et al. Tyrosine kinase inhibitors gefitinib, lapatinib and sorafenib induce rapid functional alterations in breast cancer cells. Curr Cancer Drug Targets. 2010;10:422–31. 14. Wei L, Huang N, Yang L, Zheng DY, Cui YZ, Li AM, Lu¨ CW, Zheng H, Luo RC. Sorafenib reverses multidrug resistance of hepatoma cells in vitro. Nan Fang Yi Ke Da Xue Xue Bao. 2009;29(5):1016–9, 1023. 15. Chen XP, Wang Q, Guan J, Huang ZY, Zhang WG, Zhang BX. Reversing multidrug resistance by RNA interference through the suppression of MDR1 gene in human hepatoma cells. World J Gastroenterol. 2006;12(21):3332–7. 16. Du Y, Su T, Zhao L, Tan X, Chang W, Zhang H, Cao G. Associations of polymorphisms in DNA repair genes and MDR1 gene with chemotherapy response and survival of non-small cell lung cancer. PLoS One. 2014;9(6):e99843. 17. Chen J, Ding Z, Peng Y, Pan F, Li J, Zou L, Zhang Y, Liang H. HIF-1a inhibition reverses multidrug resistance in colon cancer cells via downregulation of MDR1/P-glycoprotein. PLoS One. 2014;9(6):e98882. 18. He Q, Zhang G, Hou D, et al. Overexpression of sorcin results in multidrug resistance in gastric cancer cells with up-regulation of P-gp. Oncol Rep. 2011;25(1):237–43. 19. Shi H, Lu D, Shu Y, et al. Expression of multidrug-resistancerelated proteins P-glycoprotein, glutathione-S-transferases, topoisomerase-II and lung resistance protein in primary gastric cardiac adenocarcinoma. Cancer Invest. 2008;26(4):344–51. 20. Woehlecke H, Pohl A, Alder-Baerens N, et al. Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2). Biochem J. 2003;376(Pt 2):489–95. 21. Fan K, Fan D, Cheng LF, et al. Expression of multidrug resistancerelated markers in gastric cancer. Anticancer Res. 2000;20(6C): 4809–14. 22. Kim C, Lee JL, Choi YH, et al. Phase I dose-finding study of sorafenib in combination with capecitabine and cisplatin as a first-line treatment in patients with advanced gastric cancer. Invest New Drugs. 2012;30(1):306–15. 23. Sun W, Powell M, O’Dwyer PJ, et al. Phase II study of sorafenib in combination with docetaxel and cisplatin in the treatment of metastatic or advanced gastric and gastroesophageal junction adenocarcinoma: ECOG 5203. J Clin Oncol. 2010;28(18):2947–51. 24. Bang YJ, Kang YK, Kang WK, et al. Phase II study of sunitinib as second-line treatment for advanced gastric cancer. Invest New Drugs. 2011;29(6):L1449–58. 25. Moehler M, Mueller A, Hartmann JT, et al. An open-label, multicentre biomarker-oriented AIO phase II trial of sunitinib for patients with chemo-refractory advanced gastric cancer. Eur J Cancer. 2011;47(10):1511–20.